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Loss of Ccbe1 affects cardiac-specification and cardiomyocyte differentiation in mouse embryonic stem cells.

ABSTRACT: Understanding the molecular pathways regulating cardiogenesis is crucial for the early diagnosis of heart diseases and improvement of cardiovascular disease. During normal mammalian cardiac development, collagen and calcium-binding EGF domain-1 (Ccbe1) is expressed in the first and second heart field progenitors as well as in the proepicardium, but its role in early cardiac commitment remains unknown. Here we demonstrate that during mouse embryonic stem cell (ESC) differentiation Ccbe1 is upregulated upon emergence of Isl1- and Nkx2.5- positive cardiac progenitors. Ccbe1 is markedly enriched in Isl1-positive cardiac progenitors isolated from ESCs differentiating in vitro or embryonic hearts developing in vivo. Disruption of Ccbe1 activity by shRNA knockdown or blockade with a neutralizing antibody results in impaired differentiation of embryonic stem cells along the cardiac mesoderm lineage resulting in a decreased expression of mature cardiomyocyte markers. In addition, knockdown of Ccbe1 leads to smaller embryoid bodies. Collectively, our results show that CCBE1 is essential for the commitment of cardiac mesoderm and consequently, for the formation of cardiac myocytes in differentiating mouse ESCs.

SUBMITTER: Bover O

PROVIDER: S-EPMC6169972 | biostudies-literature |

REPOSITORIES: biostudies-literature

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1,5-Disubstituted benzimidazoles that direct cardiomyocyte differentiation from mouse embryonic stem cells.

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Project description:AIM: To investigate the effects of the cardiotonic steroid, ouabain, on cardiac differentiation of murine embyronic stem cells (mESCs). METHODS: Cardiac differentiation of murine ESCs was enhanced by standard hanging drop method in the presence of ouabain (20 ?mol/L) for 7 d. The dissociated ES derived cardiomyocytes were examined by flow cytometry, RT-PCR and confocal calcium imaging. RESULTS: Compared with control, mESCs treated with ouabain (20 ?mol/L) yielded a significantly higher percentage of cardiomyocytes, and significantly increased expression of a panel of cardiac markers including Nkx 2.5, ?-MHC, and ?-MHC. The ?1 and 2- isoforms Na(+)/K(+)-ATPase, on which ouabain acted, were also increased in mESCs during differentiation. Among the three MAPKs involved in the cardiac hypertrophy pathway, ouabain enhanced ERK1/2 activation. Blockage of the Erk1/2 pathway by U0126 (10 ?mol/L) inhibited cardiac differentiation while ouabain (20 ?mol/L) rescued the effect. Interestingly, the expression of calcium handling proteins, including ryanodine receptor (RyR2) and sacroplasmic recticulum Ca(2+) ATPase (SERCA2a) was also upregulated in ouabain-treated mESCs. ESC-derived cardiomyocyes (CM) treated with ouabain appeared to have more mature calcium handling. As demonstrated by confocal Ca(2+) imaging, cardiomyocytes isolated from ouabain-treated mESCs exhibited higher maximum upstroke velocity (P<0.01) and maximum decay velocity (P<0.05), as well as a higher amplitude of caffeine induced Ca(2+) transient (P<0.05), suggesting more mature sarcoplasmic reticulum (SR). CONCLUSION: Ouabain induces cardiac differentiation and maturation of mESC-derived cardiomyocytes via activation of Erk1/2 and more mature SR for calcium handling.

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CITED2 Cooperates with ISL1 and Promotes Cardiac Differentiation of Mouse Embryonic Stem Cells.

Project description:The transcriptional regulator CITED2 is essential for heart development. Here, we investigated the role of CITED2 in the specification of cardiac cell fate from mouse embryonic stem cells (ESC). The overexpression of CITED2 in undifferentiated ESC was sufficient to promote cardiac cell emergence upon differentiation. Conversely, the depletion of Cited2 at the onset of differentiation resulted in a decline of ESC ability to generate cardiac cells. Moreover, loss of Cited2 expression impairs the expression of early mesoderm markers and cardiogenic transcription factors (Isl1, Gata4, Tbx5). The cardiogenic defects in Cited2-depleted cells were rescued by treatment with recombinant CITED2 protein. We showed that Cited2 expression is enriched in cardiac progenitors either derived from ESC or mouse embryonic hearts. Finally, we demonstrated that CITED2 and ISL1 proteins interact physically and cooperate to promote ESC differentiation toward cardiomyocytes. Collectively, our results show that Cited2 plays a pivotal role in cardiac commitment of ESC.

| S-EPMC5161512 | biostudies-literature

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